CoUege  of  ^fipsiciansi  anli  burgeons 


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EOGcWICK.  Frederic  S.Le 

Columbia  Uuivtr 
K'«w  York, 


[From  Technology  Quarterly,  Vol.  VI,  No.  2,  July,  1893.] 


On  Methods  Used  and  Results  Obtained 


IN   MAKING 


germicidal-efficiency  tests 


DISINFECTANT 


FOR   USE  IN 


RAILWAY    SANITATION. 


By  WILLIAM  T.  SEDGWICK,  Ph.D. 


BOSTON : 

Beacon  Press;  Thomas  Todd,  Printer,  7-A  Beacon  Street, 

_j 1899. — - 1_^ 

f  From  tho  j 

Biological  Laboratory, 

MASSACHUSETTS  INSTITUTE 


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ON  METHODS  USED  AND  RESULTS  OBTAINED  IN 
MAKING  GERMICIDAL-EFFICIENCY  TESTS  OF  A 
DISINFECTANT  FOR  USE  IN  RAIL  WA  V  S ANITA  TION. 

By  WILLIAM   T.   SEDGWICK,   Ph.D. 
Received  August  18,  1893. 

The  arrival  of  Asiatic  cholera  in  the  harbor  of  New  York  in  the 
autumn  of  1892  directed  general  attention  to  matters  of  public  san- 
itation. The  disease  was  brought  upon  steamships,  and  much  of  the 
alarm  that  was  felt  was  due  to  the  knowledge  that  cholera  might  be 
carried  all  over  the  land  by  the  railroads.  The  importance  of  railway 
sanitation  had  long  been  known  to  experts,  and  the  presence  of 
cholera  at  our  doors  served  to  inform  the  public.  Fortunately,  in 
1892,  the  disease  was  confined  to  New  York. 

Recognizing  the  probability  of  a  recurrence  of  the  danger  and  the 
possibility  of  a  more  serious  invasion  in  1893,  and  anxious  to  be  ready 
for  any  emergency,  the  officers  of  the  Pennsylvania  Railroad  Company 
determined  to  have  thoroughly  tested,  with  respect  to  its  efficiency  or 
germicidal  power,  a  disinfectant  of  their  own  manufacture,  which, 
since  1885,  they  have  been  using  in  their  cars,  closets,  urinals,  pas- 
senger stations,  freight-houses,  shops,  etc.,  or  wherever  a  liquid  disin- 
fectant is  applicable  upon  a  railway  system.  The  extent  and  public 
importance  of  such  disinfection  may  be  realized  from  the  fact  that  this 
company  operates  altogether  nearly  8,000  miles  of  railway.  It  forms 
one  of  the  principal  avenues  of  communication  between  the  principal 
seaports  of  the  United  States  and  the  great  West.  It  carries  over  its 
lines  yearly  many  thousands  of  immigrants,  and  directly  connects  one 
with  another  the  largest  cities  in  the  United  States.  It  is  therefore 
held  to  be  one  of  the  most  important  railway  systems  in  America ;  and 
it  has  a  reputation  for  careful  and  scientific  administration. 

I  am  informed  by  Dr.  C.  B.  Dudley,  Chief  of  its  Chemical  Depart- 
ment, that  chemical  studies  on  commercial  disinfectants  by  this  road 
began  as  early  as  1880,  and  in   1884  these  studies  culminated  in  the 


thorough  examination  of  some  twenty-six  commercial  disinfectants. 
In  view  of  the  multiplicity  of  materials  offered  in  the  market  as  dis- 
infectants, and  the  very  high  price  charged  for  many  worthless  com- 
pounds, it  was  decided  to  embody  the  results  of  all  these  studies 
in  a  specification  for  a  disinfectant  to  be  made  for  the  use  of  the 
road.  This  specification  was  issued  in  February,  1885.  A  few 
years  later,  when  the  work  of  the  Committee  on  Disinfectants  of  the 
American  Public  Health  Association  became  available,  this  specifica- 
tion was  revised  accordingly,  and  the  so-called  P.  R.  R.  Standard  Dis- 
infectant was  brought  up  to  date. 

The  disinfectant  submitted  to  me  for  tests  of  its  germicidal  power 
was  said  to  consist  of  a  concentrated  solution  of  chloride  of  zinc, 
chloride  of  copper,  and  bichloride  of  mercury,  with  a  few  drops  of 
terebene.  The  terebene  was  added  as  a  telltale,  to  inform  the  officers 
whether  the  disinfectant  had  been  faithfully  used,  rather  than  as  a 
disinfectant.  I  was  also  informed  that  the  disinfectant  contains  one 
part  in  five  hundred  of  bichloride  of  mercury  and  a  little  over  45  per 
cent,  of  chloride  of  zinc.  The  chloride  of  copper  was  added  because 
a  little  copper  salt  was  desired  to  fix  sulphuretted  hydrogen,  and 
the  chloride  of  zinc  solution  was  made  very  strong  on  account  of  the 
well-known  deodorizing  properties  of  this  material. 

I  am  also  informed  by  Dr.  Dudley  that  this  disinfectant  is  made 
by  the  railroad  company,  for  its  own  use,  under  the  supervision  of 
the  officers  of  its  laboratory.  Old  battery  zincs  which  are  no  longer 
useful  in  the  batteries,  muriatic  acid,  sulphate  of  copper,  common  salt, 
and  bichloride  of  mercury  are  used  in  its  manufacture.  The  actual 
cost  of  the  disinfecting  material  in  an  eight-ounce  bottle  is  about  two 
and  one  half  cents.  The  material  is  distributed  to  the  service  in 
wooden  boxes,  holding  twenty-four  bottles  each,  and  in  barrels.  If 
these  boxes  and  bottles  are  returned,  the  real  cost  to  the  different 
parts  of  the  service  is  the  figure  given  above.  If  the  bottles  are 
broken  and  the  boxes  destroyed  the  cost  is  somewhat  greater.  In 
barrels  which  are  not  returned  the  cost  is  somewhat  less  than  in 
bottles,  no  labor  being  required  for  bottling.  There  were  made  and 
used  on  the  whole  Pennsylvania  railway  system  during  the  year  1892 
366  gross  of  eight-ounce  bottles  and  69  fifty-gallon  barrels. 

The  specifications  to  which  the  disinfectant  must  conform  are 
given  as  follows  : 

"  Disinfectant  for  the  use  of  the  Pennsylvania  Railroad  Company 


will  consist  of  a  neutral  solution  of  the  normal  chlorides  of  copper  and 
zinc  and  mercuric  chloride  put  up  in  eight  (8)  ounce  bottles  and  se- 
curely corked.  Each  bottle  should  contain  2,400  grains  of  zincic  chlo- 
ride, 120  grains  of  cupric  chloride,  io.$  grains  of  mercuric  chloride, 
and  ten  drops  of  a  mixture  of  equal  parts  of  terebene  and  turpentine." 
The  bottles  must  have  pasted  upon  them  the  following  "  Directions 
for  Use,"  a  perusal  of  which  will  convey  a  good  idea  of  the  scope 
and  the  importance  to  the  community  of  efficient  railway  sanitation  : 

DIRECTIONS    FOR    USE. 

For  Use  on  Cars. — Empty  the  contents  of  this  bottle  into  about  one  quart  of  water. 
With  a  sponge  or  waste,  wash  with  the  liquid  all  parts  of  the  urinal,  seat,  and  hopper,  and 
the  floor  adjacent  to  them  in  the  closet,  allowing  some  of  the  liquid  to  run  down  the 
urinal  pipes.  The  same  solution  should  be  used  on  the  floors  and  woodwork  of  the  whole 
car  whenever  washing  with  disinfectant  is  necessary.  The  carpets  and  plush  of  an  infected  car 
should  not  be  treated  with  disinfectant,  but  should  be  fumigated  instead. 

For  Cleaning  Privies,  Closets,  etc.  —  Empty  the  contents  of  this  bottle  into  about 
one  quart  of  water,  and  use  as  above.  The  disinfectant  acts  slowly  on  lead,  tin,  copper, 
brass,  iron,  etc.,  and  it  is  not  advisable  to  allow  it  to  stand  in  the  basins  or  traps.  In  the 
vault,  use  the  disinfectant  full  strength,  not  less  than  half  a  dozen  bottles  at  once,  but  it  is 
better  to  disinfect  privy  vaults  with  crushed  sulphate  of  copper,  or  chloride  of  lime,  sprinkled 
freely  over  the  mass. 

For  Removing  Bad  Odors.  —  Empty  the  contents  of  this  bottle  into  about  one  quart 
of  water,  and  sprinkle  the  liquid  in  the  place  from  which  the  odors  arise;  also  saturate  a 
towel,  or  other  fibrous  substance,  with  the  liquid  and  hang  it  in  the  place  from  which  it  is 
desired  to  remove  the  odors.  For  use  on  towels,  etc.,  a  disinfectant  without  color  can  be 
furnished  if  desired. 

In  Cases  of  Diphtheria,  Scarlet,  Typhoid,  and  Typhus  Fevers,  Cholera,  Dys- 
entery, Consumption,  etc.  —  Place  a  little  of  the  disinfectant,  full  strength,  in  the  vessel 
which  receives  the  excrement,  or  vomit,  or  throat  discharge,  and  pour  more  of  the  disin- 
fectant on  the  discharge  after  it  has  been  received  in  the  vessel.  If  the  discharge  soils  the 
floor  or  furniture  wash  these  soiled  spots  with  the  disinfectant  mixed  with  water  as  above. 
Soiled  bed  and  body  linen,  rags,  or  handkerchiefs  used  to  receive  discharges,  and  soiled 
clothing  should  be  burned,  or  soaked  in  disinfectant  diluted  as  above,  for  four  hours  and 
then  rinsed  with  water. 

For  the  Dead,  where  Disinfectant  is  Necessary. —  W^rap  the  body  in  a  sheet 
thoroughly  saturated  with  the  disinfectant  full  strength. 

For  Attendants  on  the  Sick.  —  Empty  the  contents  of  this  bottle  into  a  pint  of 
water,  and  wash  the  hands  and  other  portions  of  the  body,  if  sailed,  with  it,  rinsing  off  with 
clean  water.     In  sick  rooms  and  hospital  wards,  wash  all  surfaces  with  tne  same  solution. 

2i^="This  disinfectant,  when  mixed  with  water,  as  described,  will  not  injure  the  hands, 
but  should  not  get  into  the  mouth. 

As  has  been-  stated  above,  in  view  of  the  probability  of  a  fresh 
invasion  of  cholera  in  1893,  the  officers  of  the  road  determined  to  be 
as  fully  prepared  for   it   as  possible,  and  accordingly,    in   December, 


1892,  Mr.  Theodore  N.  Ely,  then  Superintendent,  now  Chief,  of  Motive 
?ower,  desired  me  to  make  a  careful  series  of  tests  to  determine,  in 
the  most  rigid  manner,  the  precise  germicidal  value  of  their  standard 
disinfectant,  and  to  suggest  to  them,  if  I  could,  how  its  efficiency 
might  be  improved. 

In  view  of  the  fact  that  the  composition  of  the  disinfectant  is 
well  known,  that  it  is  not  for  sale,  except  to  its  employees  or  where 
the  interests  of  the  road  are  directly  involved,  and  is  not  in  any  sense 
a  proprietary  preparation  and,  above  all,  impressed  with  the  im- 
portance of  the  inquiry  to  the  public  welfare,  I  consented  to  under- 
take the  investigation,  although  with  reluctance  because  of  the  peculiar 
difficulties  involved  in  all  efficiency  tests  upon  germicides.  I  was  for- 
tunate, however,  in  securing  throughout  the  investigation  the  aid  of 
Mr.  Albert  P.  Mathews,  S.B.,  Assistant  in  Biology  at  the  Massachu- 
setts Institute  of  Technology,  to  whose  skill  and  devotion  the  work 
owes  much  of  whatever  value  it  may  possess. 

In  the  present  state  of  opinion  regarding  the  active  cause  of 
infectious  disease,  which  holds  that  this  is  invariably  due  to  specific 
microorganisms,  it  becomes  simple  in  theory  to  determine  the  value 
of  any  germicide  or  antiseptic.  It  seems,  at  first  sight,  easy  enough 
to  test  the  efficiency  of  a  disinfectant  by  simply  observing  under 
various  conditions  its  germicidal  power.  But  in  arranging  the  details 
of  the  experiments  difficulties  arise  which  are  hard  to  overcome. 

It  might  be  supposed  that  if  we  exposed  known  numbers  of  spe- 
cific disease  germs  to  the  action  of  a  fixed  amount  of  the  disinfectant  for 
a  definite  period,  and  then  made  cultures  from  the  mixture,  we  should 
readily  settle  the  efficiency  of  the  disinfectant  at  the  given  strength. 
But  this  is  by  no  means  the  case ;  for,  unless  great  care  be  taken  to- 
prevent  it,  we  may  carry  over  with  the  test  sample  of  the  mixture 
taken  for  planting  enough  of  the  disinfectant  to  inhibit  later  the  de- 
velopment of  the  still-living  germs.  Errors  of  this  kind  are  partic- 
ularly insidious  and  very  difficult  to  avoid.  Again,  there  is  reason  to 
believe  that  cultures  of  the  germs  submitted  to  examination  vary  in 
their  endurance  considerably,  particularly  if  these  are  of  kinds  which 
produce  spores.  As  in  the  higher  plants  some  fields  of  corn  or  oats 
are  strong  and  vigorous  while  others  are  weak  and  sickly,  so  here  it  is 
important  to  know  that  the  cultures  or  crops  of  bacteria  with  which 
we  are  dealing  are  fairly  representative  of  those  which  devastate  the 
human  body. 


5 

The  problem  in  this  case  was  strictly  one  of  germicidal  and  not 
merely  antiseptic  properties.  For  the  preservation  of  food  the  prob- 
lem is  often  one  of  antisepsis,  but  into  disinfection  this  milder 
problem  does  not  enter.  The  question  is  one  of  destruction,  not  in- 
hibition, and  may  be  stated  thus  :  How  efficient  is  the  disinfectant  as 
a  destroyer  of  the  germs  of  infectious  disease  ? 

The  methods  used  in  the  examinations  of  the  germicidal  power  of 
■disinfectants  have  been  almost  as  numerous  as  the  observers,  and  the 
results  obtained  have  varied  widely.  Koch  has  employed  a  silk  thread 
dipped  into  the  culture  and  then  exposed  to  the  action  of  the  disin- 
fectant. The  thread  was  afterwards  washed  with  sterile  distilled 
water,  and  then  planted  on  the  surface  of  the  plated  medium  or  else 
sown  in  bouillon.  Such  a  method  is  open  to  serious  objection,  as  it 
is  difficult  to  wash  away  all  the  disinfectant  clinging  to  the  thread 
and  the  slight  amount  left  may  continue  to  act  antiseptically,  and 
lead  to  erroneous  conclusions. 

Sternberg,  in  this  country,  has  proceeded  in  a  different  manner. 
He  exposes  the  broken-down  beef  tea,  or  pure  culture  of  the  bacteria 
in  bouillon,  to  a  known  strength  of  the  germicide  for  a  given  length  of 
time,  after  which  he  transfers  by  peculiar  flasks  a  certain  small  amount 
to  a  large  amount  of  fresh  alkaline  bouillon  and  allows  incubation  to 
proceed.  The  objection  to  this  method  is  that  he  inevitably  intro- 
duces into  the  second  culture  medium  an  uncertain  small  amount  of 
the  disinfectant.  The  methods  of  Koch  and  Sternberg  are  both,  at 
least  in  theory,  somewhat  untrustworthy,  for  the  reason  that  the 
action  of  the  germicide  cannot  be  checked  at  once  and  because  each 
introduces  into  his  control  medium  some  of  the  germicide. 

It  is  self-evident  that  in  testing  any  germicide  it  is  necessary  to 
stop  its  action  quickly  or  to  remove  it  wholly  from  the  test  medium. 
Only  when  we  accomplish  this  can  we  hope  to  be  sure  of  the  results. 

In  solving  the  present  problem  we  were  guided  by  the  work  of 
those  experimenters  who  had  also  recognized  the  necessity  of  freeing 
the  bacteria  after  their  exposure  for  the  given  time  from  the  possible 
antiseptic  influences  of  small  amounts  of  the  germicide  thus  carried 
over.  Two  reagents  have  hitherto  been  used  for  this  purpose  for 
antiseptics  containing  corrosive  sublimate,  namely,  ammonium  sulphide 
and  hydrogen  sulphide.  Our  problem,  however,  was  not  so  simple. 
In  the  first  place,  it  was  not  only  necessary  to  test  the  antiseptic 
effect  of  the  precipitant  upon  every  specific  germ  to  assure  ourselves 


that  it  was  not  itself  more  or  less  antiseptic  ;  but  in  the  second  place, 
we  were  here  not  merely  working  with  a  dilute  solution  of  the  subli- 
mate, but  also  with  a  strong  solution  of  zincic  and  cupric  chlorides^ 
and  if  either  of  the  precipitants  mentioned  were  used  there  would  be 
formed  a  heavy,  fiocculent  precipitate  likely  to  gather  around  the  germs 
and  drag  them  down,  thus  leading  to  serious  error.  Hence  we  were 
compelled  to  run  control  experiments  with  pure  water  in  many  cases,, 
and  sometimes  with  other  precipitants,  while  at  the  same  time  carefully 
testing  the  antiseptic  action  of  the  precipitant  itself. 

As  already  stated,  we  endeavored  to  eliminate  the  various  sources 
of  error  that  had  hitherto  been  recognized.  The  first  question  was 
that  of  the  cultures  themselves,  and  only  the  freshest  cultures  of 
pathogenic  germs  were  used,  since  it  is  probable  that  such  cultures 
are  most  nearly  normal.  We  also  satisfied  ourselves  that  each  culture 
was  in  a  vigorous  state,  as  indicated  by  the  rapidity  of  its  growth  or 
the  manner  in  which  it  formed  spores. 

The  cultures  of  the  specific  bacteria  used  were  subjected  to  rigid 
differential  tests  in  order  to  establish  the  fact  that  they  were  really 
the  germs  in  question.  The  culture  of  typhoid  fever  bacilli  had 
been  isolated  not  long  before  from  the  spleen  of  a  woman  dead  of 
typhoid  fever.  The  culture  of  anthrax  was  comparatively  fresh,  having 
been  obtained  from  a  patient  in  the  Boston  City  Hospital.  The  cul- 
ture of  Staphylococcus  pyogenes  am-eus  was  derived  from  one  isolated 
a  few  days  previous  by  Dr.  W.  T.  Councilman,  Pathologist  to  the  Bos- 
ton City  Hospital.  The  culture  of  the  Streptococcus  of  diphtheria  was 
also  derived  from  one  isolated  by  Dr.  Councilman  a  few  days  before  it 
was  used  in  these  experiments.  The  culture  of  the  cholera  Spirilhivt 
of  Koch  was  derived  from  one  isolated  by  Dr.  Dunham,  of  New  York, 
from  a  case  of  Asiatic  cholera  there  in  September  last.  The  culture  of 
Bacillus  subtilis  had  been  in  the  laboratory  for  some  time,  but,  as  was 
proved  by  the  resistant  properties  of  its  spores  when  subjected  to 
heat,  it  was  in  a  most  vigorous  condition.  We  may  then  safely  con- 
clude that  the  cultures  employed  in  the  tests  were  in  a  fresh  and  vig- 
orous condition. 

It  is  generally  believed  that  mercury  forms  a  more  or  less  stable 
compound  with  albumen,  though  whether  this  compound  is  or  is  not 
soluble,  and  whether  it  is  or  is  not  effective  in  making  the  germicide 
powerful,  is  a  matter  of  dispute.  Vaughan  contends  that  it  is  very 
poisonous,  while  others  hold  that  such  an  envelope  forming  about  the 


bodies  of  the  bacteria  protects  them  from  the  further  action  of  the 
germicide.  How  far  these  ideas  are  true,  or  how  far  they  are  im- 
portant if  true,  is  uncertain. 

In  the  actual  investigations  we  introduced  a  known  amount  of  the 
fluid  holding  the  germs  in  suspension  into  a  known  amount  of  the  dis- 
infectant, and  at  the  end  of  a  given  interval  transferred  a  small  known 
amount  of  this  mixture  to  a  large  amount  of  dilute  ammonium  sulphide 
which  was  used  as  the  inhibitor,  sometimes  making  control  experiments 
with  stannous  chloride  and  at  other  times  with  water  in  the  place  of 
the  ammonium  sulphide.  We  used  ammonium  sulphide  in  preference 
to  hydrogen  sulphide  as  inhibitor,  for  the  reason  that  the  solution  of 
the  germicide  was  found  to  be  decidedly  acid,  and  thus  we  were  forced 
to  neutralize  the  acid  at  the  same  time  that  the  mercury  was  neu- 
tralized. The  use  of  any  sulphide  we  adopted  with  hesitation,  owing 
to  the  formation  of  a  heavy  flocculent  precipitate  ;  but  by  making 
control  tests  with  stannous  chloride,  which  simply  neutralized  the 
mercury  and  did  not  affect  the  other  chlorides,  and  by  using  water  in 
the  place  of  the  sulphide,  we  came  to  the  conclusion  that,  in  spite  of 
this  danger,  the  ammonium  sulphide  was  the  best  inhibitor  we  could 
use,  although  it  may  be  that  further  experiment  would  show  the 
stannous  chloride  to  be  equally  valuable. 

We  thus  succeeded  in  stopping  the  action  of  the  disinfectant 
upon  its  arrival  in  the  second  flask,  and  by  employing  large  dilutions 
further  reduced  the  possibility  of  the  contamination  of  the  culture 
medium  with  an  antiseptic  salt.  It  will  be  noticed,  also,  in  examining 
the  detailed  history  of  the  experiments,  that  through  planting  a  con- 
trol from  the  second  flask  directly  into  a  large  amount  of  bouillon 
we  checked  our  final  results  and  escaped  the  danger  of  losing  the 
germs  through  too  great  dilutions. 

In  all  experiments  controls  were  made  into  bouillon  to  determine 
the  initial  sterility  of  the  dilution  flask ;  a  sample  was  also  planted 
from  the  precipitating  flask  to  make  sure  that  the  bacteria  were  not 
lost  in  passing  from  the  precipitating  or  inhibition  flask  to  the  third 
or  dilution  flask ;  and  finally,  controls  were  made  to  discover  how 
many  bacteria  would  have  appeared  in  a  cubic  centimeter  of  the  third 
flask  had  the  disinfectant  been  water.  We  have  thus  had  a  constant 
check  on  all  results,  and  feel  confident  that  error  has  as  far  as  possible 
been  eliminated. 

In  order,  however,  to  guard  against  all  possible  error,  when  the  end 


8 

of  the  experiment  was  reached  and  the  limit  of  the  resistance  of  the 
bacterium  had  been  determined  by  means  of  the  precipitation  methods, 
two  other  methods  were  used  in  order  to  check  the  results.  One  of 
these  was  that  recommended  by  Sternberg,  namely,  exposing  the  cul- 
ture to  the  germicide  for  a  certain  length  of  time  and  then  transferring 
a  sm.all  quantity  of  the  mixture  —  as  much  as  would  cling  to  a  platinum 
needle  —  into  a  flask  of  bouillon  and  allowing  the  latter  to  grow  at 
optimum  temperature.  The  other  method  was  to  transfer  a  platinum 
loopful  of  the  mixed  liquid  culture  and  germicide  to  a  weak  solution 
of  ammonium  sulphide  and  then  pour  the  whole  of  this  into  a  large 
flask  of  bouillon.  In  the  latter  method  we  escaped  both  the  diffi- 
culty of  introducing  the  corrosive  sublimate  into  the  culture  and  the 
danger  of  great  dilutions. 

The  precise  method  of  procedure  ^  was  this  :  Bouillon  cultures  of 
the  species  to  be  tested  were  grown  for  two  days  in  the  incubator  at 
37.5°C.  Two  cubic  centimeters  of  such  a  culture,  after  prolonged  shak- 
ing, and  after  microscopical  and  macroscopical  examination  had  deter- 
mined that  it  was  in  good  condition,  were  transferred  to  a  60  cc.  sterile 
Erlenmeyer  flask,  and  to  them  1 3  cc.  of  sterile  distilled  water  were 
added.  An  equal  volume,  or  15  cc,  of  the  germicide  was  next  added, 
and  the  time  of  exposure  was  determined  by  means  of  a  stop  watch. 
The  flask  here  used  may  be  called  the  action  flask,  and  the  time  of 
exposure  the  action  period.  Towards  the  end  of  the  action  period 
one  cubic  centimeter  was  rapidly  transferred  to  500  cc.  of  dilute  solu- 
tion of  ammonium  sulphide,  or  stannous  chloride,  and  well  shaken  for 
thirty  seconds,  the  precipitate  not  being  allowed  to  settle.  At  the 
end  of  the  thirty  seconds  one  cubic  centimeter  from  this  flask,  which 
may  be  called  the  inhibition  flask,  was  transferred  to  a  flask  contain- 
ing 250  cc.  of  sterile  distilled  water;  the  last  flask  may  be  called  the 
dilution  flask.  Immediately  after  this  two  flasks,  containing  each 
25  cc.  of  bouillon,  were  planted,  each  with  .3  cc,  from  the  inhibition 
flask.  This  was  done  in  order  to  have  a  dilution  control.  At  the  end 
of  a  couple  of  minutes  two  or  three  tubes  of  melted  agar  were  sown 
with  one  cubic  centimeter  each  from  the  mixture  in  the  third  or  dilu- 
tion flask,  and  immediately  plated.  All  plates  and  flasks  were  then 
placed  in  the   incubator  at  37.5°C. 

Immediately  preceding    the  experiment    one    cubic  centimeter    of 


'  In  formulating  a  definite  mode  of  procedure  we  were  guided,  in  part,  by  experiments 
made  in  this  laboratory  upon  peroxide  of  hydrogen  by  the  late  George  V.   McLauthlin. 


water  from  the  dilution  flask  was  planted  in  bouillon  in  order  to  de- 
termine its  sterility.  This  precaution  was  found  to  be  necessary  when 
working  with  the  spores  of  B.  sicbtilis  and  B.  anthracis,  both  of  which 
proved  to  be  very  resistant  to  ordinary  sterilization  methods. 

Immediately  following  each  series  of  experiments  a  control  experi- 
ment was  made  as  follows  :  Two  cubic  centimeters  of  the  bouillon 
culture  used  in  testing  were  diluted  with  28  cc.  of  water,  and  one 
cubic  centimeter  of  this  diluted  culture  carried  into  500  cc.  of  am- 
monium sulphide  in  the  inhibition  flask.  After  thirty  seconds  one 
cubic  centimeter  from  the  mixture  in  the  inhibition  flask  was  carried 
to  the  dilution  flask,  containing  250  cc.  of  sterile  water,  and  from  the 
last  flask  agar  plates  were  made  by  sowing  in  each  tube  of  melted 
agar  one  cubic  centimeter  of  the  liquid  and  then  plating  the  mass. 
Two  other  controls  were  obtained  for  each  species  by  substituting  for 
ammonium  sulphide  in  the  inhibition  flask  stannous  chloride  in  the 
one  case  and  distilled  water  in  the  other,  and  making  the  dilutions 
the  sg.me  as  in  the  principal  testing  experiments. 

This  routine  was  repeated  for  the  different  action  periods  until  it 
was  found  that  the  germs  were  killed.  When  this  knowledge  had 
been  obtained  still  another  control  was  employed,  namely,  Sternberg's 
method  and  the  modification  of  it  already  described. 

It  will  thus  appear  that  all  results  obtained  were  carefully  checked, 
and  all  tests  finally  made  in  duplicate  or  triplicate.  After  the  actual 
theoretical  value  of  the  germicide  had  been  thus  established,  its  ac- 
tual value  in  practice  was  proved  by  its  action  on  a  typhoid  fever  stool. 

A     DETAILED     ACCOUNT     OF    THE     EXPERIMENTS     MADE     TO     DETERMINE 
THE    GERMICIDAL    EFFICIENCY    OF    THE    DISINFECTANT. 

A.     Experiments  upon  the   Typhoid  Fever  Bacillus  of  E berth. 

Inasmuch  as  the  tests  with  this  bacillus  were  the  first  undertaken, 
it  was  necessary  to  develop  the  methods  to  be  used  in  testing.  The 
first  few  experiments  were  therefore  intended  to  determine  whether 
the  stannous  chloride  or  the  ammonium  sulphide  were  objectionable 
because  of  being  germicides  in  themselves,  and  whether  either  method 
was  superior  to  that  of  simple  dilution  in  water. 

Experiment  i,  when  compared  with  Experiment  2,  indicated  that 
the  method  of  inhibition  by  precipitating  with  stannous  chloride,  was 
superior  to  that  of  diluting  with  water.     Experiment  i,  in  which  the 


lO 

chloride  was  used,  indicated  that  the  germs  were  still  alive  after  one 
minute's  exposure,  while  Experiment  2  showed  that  all  had  been  killed. 
If,  then,  we  had  relied  upon  the  method  of  simple  dilution  we  should 
have  been  led  into  error  at  the  start. 

EXPERIMENT    I. 

I  volume  of  disinfectant  diluted  with  3  volumes  of  water. 

I  cc.  of  50-days-old  bouillon  culture  of  Eberth  bacilli  introduced  into  250  cc.  of  this 
diluted  disinfectant  and  exposed  for  one  minute. 

I  cc.  of  the  mixture  of  disinfectant  and  culture  then  carried  to  500  cc.  of  dilute,  weakly 
acid  stannous  chloride,  and  left  for  35  seconds. 

I  cc.  of  liquid  from  the  stannous  chloride  flask  then  mixed  with  500  cc.  of  water  and 
well  shaken. 

From  the  last  flask  five  agar  plates  were  planted  with  one  cc.  each. 

Result :  Average  number  of  colonies  per  cc.  of  last  flask,  3. 

EXPERIMENT   2. 

Same  conditions  as  in  preceding  experiment.  Disinfectant  same  strength  and  in  same 
amount,  and  the  same  amount  of  the  same  culture  used. 

Exposure  to  the  disinfectant,  one  minute. 

1  cc.  of  mixture  of  culture  and  disinfectant  then  carried  to  500  cc.  of  sterile  water  and 
well  shaken  for  35  seconds. 

I  cc.  of  this  fluid  then  carried  to  500  cc.  of  sterile  water. 

Five  agar  plates  were  planted  with  i  cc.  of  the  last  flask. 

Result :  All  the  plates  were  sterile. 

These  experiments  having  indicated  the  value  of  the  stannous 
chloride  as  an  inhibitor,  we  next  proceeded  to  determine  that  of  the 
ammonium  sulphide. 

EXPERIMENT   3. 

2^-days-old  bouillon  culture  of  Eberth  bacilli.     Grown  at  37.5°C. 

I  cc.  of  this  culture  carried  to  250  cc.  of  disinfectant  diluted  as  in  i  and  2.  Exposed 
20  seconds. 

I  cc.  of  the  disinfectant  and  culture  then  mixed  wtth  500  cc.  of  ammonium  sulphide. 
Exposed  for  20  seconds. 

I  cc.  of  this  mixture  from  Flask  2  well  washed  in  500  cc.  of  sterile  water. 

Five  agar  plates  were  then  made  from  the  last  flask,  each  plate  containing  i  cc. 

Result:  Three  plates  were  sterile ;  two  had  one  colony  each  upon  them. 

In  order  to  determine  whether  a  longer  exposure  to  the  ammonium  sulphide  would  in- 
jure the  bacteria  we  allowed  the  inhibition  flask  to  stand  for  three  minutes,  when  we  again 
diluted  one  cubic  centimeter  of  it  with  500  cc.  of  sterile  water,  and  after  shaking  planted  five 
cubic  centimeters  of  the  last  dilution  in  five  tubes  of  agar. 

Result:  Two  of  the  plates  had  one  colony  each;  one  of  them  contained  three;  and  the 
third  was  sterile. 

This  indicated  that  the  sulphide  possessed  a  very  slight  action 
upon  the  bacteria,  if  it  exerted  any  at  all.  The  slightly  greater 
numbers  of    bacteria  grown  on  these  plates  were  wholly  within   the 


II 

range  of  variation  in  exactly  similar  series  of  experiments.  Other- 
wise they  would  seem  to  indicate  that  a  longer  exposure  of  the 
bacteria  to  the   sulphide  was  advantageous. 

The  next  experiment  was  made  to  test  the  value  of  washing 
the  disinfectant  from  the  bacteria  by  water. 

EXPERIMENT   4. 

Same  conditions  and  culture  as  in  Experiment  3,  but  the  exposure  was  for  18  seconds. 
I  cc.  of  mixture  of  disinfectant  and  culture  was  well  shaken  in  500  cc.  of  sterile  water 
for  20  seconds,  Flask  2. 

I  cc.  from  Flask  2  then  shaken  in  500  cc.  of  sterile  water,  Flask  3. 

Six  agar  plates  were  planted  with  i  cc.  each  from  Flask  3. 

Result :  three  plates  sterile  ;  two  plates  had  one  colony  each  upon  them. 

The  result  of  this  experiment  would  seem  to  indicate  that  sim- 
ple dilution  was  as  effective  as  ammonium  sulphide  precipitation. 
Further  experiment  proved  that  this  conclusion  was  erroneous. 
Experiment  5  is  the  control  for  the  two  preceding,  made  in  order 
to  determine  whether  the  bacteria  had  been  lost  by  too  great 
dilution. 

EXPERIMENT    5. 

I  cc.  of  same  culture  as  in  Experiment  4  diluted  with  sterile  water  to  the  same  degree 
that  it  would  have  been  diluted  had  it  passed  through  the  action,  inhibition,  and  dilution 
flasks.     No  disinfectant  used. 

Six  agar  plates  planted  with  i  cc.  each  of  Flask  3. 

Result :  These  plates  had  an  average  of  eleven  colonies  each  upon  them. 


The  result  of  Experiment  5  was  sufficient  to  convince  us  that  the 
dilution  of  the  disinfectant  was  too  great  ;  and  thereafter,  with  the 
exception  of  the  next  control  experiment,  we  reduced  the  last  flask 
to  250  cc.  ;  reduced  the  disinfectant  volume  to  15  cc.  ;  and  employed 
two  cc.   of  the  culture  in  the  place  of  one. 

EXPERIMENT    6. 

This  was  made  to  test  the  germicidal  power  of  the  stannous  chloride. 
I  cc.  of  culture  was  carried  to  250  cc.  of  sterile  water. 

I  cc.  of  this  mixture  then  planted  in  500  cc.  of  dilute  stannous  chloride  and  left  for  20 
seconds. 

I  cc.  of  this  then  carried  to  500  cc.  of  sterile  water. 

Six  agar  plates  were  planted  each  with  i  cc.  of  last  flask. 

Result :  There  was  an  average  number  of  14  colonies  upon  each  plate. 


12 

This  result  proved  that  the  stannous  chloride  in  the  length  of 
time  the  bacteria  was  exposed  to  it,  and  in  the  strength  in  which 
it  was  used,  exerted  no  appreciable  deleterious  effect  upon  the  typhoid 
bacilli. 

Having  now  made  a  test  of  methods,  in  which  it  appeared  that 
there  was  little  advantage  in  using  stannous  chloride  in  preference 
to  ammonium  sulphide  as  inhibitor,  we  proceeded  to  determine  the 
efficiency  of  the  disinfectant.  For  this  purpose  we  employed  the 
disinfectant  diluted  to  twice  its  normal  bulk.  This  dilution  was  se- 
cured in  the  early  experiments  by  means  of  the  culture  fluid  itself. 
Later,  however,  we  diluted  with  water.  This  strength  of  the  dis- 
infectant was  adopted  as  the  standard  both  because  it  seemed  likely 
that  such  a  strength  would  be  the  one  employed  in  practice  and 
because  we  were  thereby  enabled  to  compare  its  efficiency  with  that 
of  neutral  solutions  of  mercuric  chloride  of  the  strength  i  :  i,ooo  as 
determined  by  many  experimenters. 

EXPERIMENT  7. 

Fifteen  cc.  of  a  two-days  culture  in  bouillon  of  Eberth  bacilli  were  mixed  with  15  cc.  of 
disinfectant  full  strength.  Thus  the  culture  acted  as  the  diluter  of  the  disinfectant.  The 
exposure  was  for  25  seconds. 

I  cc.  of  the  mixture  was  then  carried  to  500  cc.  of  stannous  chloride  and  shaken  for  20 
seconds. 

I  cc.  from  this  flask  was  then  transferred  to  Flask  3,  containing  250  cc.  of  sterile  water. 

Five  agar  plates  were  then  planted  from  the  last  flask  with  one  cc.  each. 

Result:  All  the  plates  were  sterile. 

EXPERIMENT   8. 

Identical  with  the  preceding,  except  that  the  culture  was  exposed  to  the  disinfectant  for 
45  seconds. 

Five  agar  plates  planted  from  the  last  flask. 
Result:  All  plates  sterile. 

EXPERIMENT   9. 

Control  of  7  and  8. 

I  cc.  of  culture  transferred  to  500  cc.  of  stannous  chloride  for  20  seconds. 

I  cc.  of  this  fluid  then  diluted  with  250  cc.  of  sterile  water. 

Two  agar  plates  planted  with  i  cc.  each  from  the  last  flask. 

Result:   2,035  colonies  upon  each  plate. 

It  was  obvious  from  the  preceding  experiments  that  twenty-five 
seconds'  exposure  sufficed  to  kill  the  bacteria.  We  now  determined 
the  minimum  length  of  time  of  exposure  necessary  to  accomplish 
this    result.      Here  we    adopted    a    slightly  different    method  of    pro- 


13 

cedure.  It  will  be  observed  that  the  disinfectant  was  diluted  in 
these  cases  with  the  culture  medium  itself.  Considering  that  this 
medium  was  of  an  albuminous  nature  and  therefore  likely  to  intro- 
duce an  element  of  uncertainty  into  the  results,  as  the  exact  solu- 
bility of  the  albuminate  of  mercury  is  a  matter  of  dispute,  and  its 
effect  upon  the  germicidal  value  of  mercuric  salts  has  also  been 
questioned,  we  freed  the  bacteria  from  this  albuminous  envelope  as 
far  as  possible  by  thoroughly  shaking  two  cubic  centimeters  of  the 
culture  in  sterile  water  sufficient  to  dilute  the  disinfectant  to  twice 
its  bulk.  Hereafter,  then,  it  will  be  understood  that  the  15  cc.  of 
disinfectant  were  diluted  with  13  cc.  of  water  and  2  cc.  of  culture 
medium.  In  this  way  the  bacteria  themselves  were  exposed  more 
directly  to  the  action  of  the  germicide. 

EXPERIMENT    lO. 

Fifteen  cc.  of  disinfectant  mixed  with  13  cc.  of  water  and  2  cc.  of  culture  of  typhoid 
bacilli.     Exposure  of  10  seconds. 

I  cc.  of  mixture  carried  to  500  cc.  of  stannous  chloride  for  20  seconds. 

I  cc.  from  this  flask,  known  as  the  inhibition  flask,  next  transferred  to  a  dilution  flask 
containing  250  cc.  of  sterile  water. 

Two  bouillon  flasks  containing  25  cc.  each  of  bouillon  were  then  sown  with  0.2  cc.  each 
from  the  inhibition  flask  (No.  2). 

Two  agar  plates  were  made  from  Flask  3. 

Result:  One  bouillon  flask  sterile ;  other  bouillon  flask  growth.  Agar  plates,  two  sterile; 
the  third  contained  two  colonies. 

EXPERIMENT    II. 

Identical  with  10,  except  that  the  exposure  to  the  disinfectant  was  for  15  seconds. 
Agar  plates  and  bouillon  flasks  planted  as  in  the  preceding  experiment. 
Result :  both  i^lates  sterile.     Both  bouillon  flasks  sterile. 

EXPERIMENT    12. 

Identical  with  lo,  except  that  the  exposure  to  the  disinfectant  was  for  20  seconds. 

Agar  plates  and  bouillon  flasks  planted  as  in  10. 

Result :  Agar  plates  were  sterile.     Bouillon  flasks  were  sterile. 

From  these  experiments  it  follows  that  the  Eberth  bacillus  of 
typhoid  fever  is  killed  by  an  exposure  to  the  disinfectant  —  the  latter 
being  diluted  to  one  half  its  original  strength  —  of  about  twelve 
seconds.  It  is  not  killed  in  ten  seconds,  but  it  is  in  fifteen.  We 
now  proceeded  to  control  this  result  by  inhibition  with  ammonium 
sulphide  in  the  place  of  the  stannous  chloride. 


EXPERIMENT   I3. 

Identical  with  11,  except  that  ammonium  sulphide  was  placed  in  the  middle  flask  in  place 
of  the  stannous  chloride.  The  culture  was  the  same  and  the  dilutions  were  to  the  same 
amount  and  of  the  same  kind. 

Exposure  of  culture  to  the  disinfectant  for  15  seconds.  Carried  to  the  sulphide  and  ex- 
posed for  30  seconds. 

Agar  plates  made  from  Flask  3,  and  bouillon  flasks  from  Flask  2. 

Result:  Agar  plates  sterile.     Bouillon  flasks  remained  sterile. 

EXPERIMENT    I4. 

This  experiment  the  same  in  all  respects  as  13,  except  that  the  exijosure  to  the  disin- 
fectant was  for  TO  seconds  only. 

Result:  Bouillon  flasks  sterile;  agar  plates  sterile. 

It  will  be  seen  that  the  inhibition  with  ammonium  sulphide  con- 
firmed the  results  of  inhibition  with  stannous  chloride.  The  only 
difference  was  that  the  latter  method  showed  some  of  the  bacteria 
to  be  still  alive  after  ten  seconds'  exposure  to  the  disinfectant,  while 
the  former  indicated  that  all  had  been  killed.  It  may  have  been 
that  in  the  former  case  the  exposure  was  for  a  second  or  two  longer 
than  in  the  latter,  as  it  was  impossible  to  work  with  an  accuracy 
within  a  second. 

The  control  experiments  for  the  above,  to  guard  that  the  bac- 
teria should  not  be  lost  from  too  great  dilution,  showed  the  last 
flask  to  contain  150  colonies  to  the  cubic  centimeter,  and  proved 
the  dilutions  to  be  safe.  We  also  made  another  control,  to  deter- 
mine that  the  stannous  choride  was  harmless.  The  plates  showed 
an  average  of  104  colonies  to  the  cubic  centimeter  —  a  result  within 
the  range  of  unavoidable  differences  in  the  number  of  bacteria  trans- 
ferred from  flask  to  flask. 

The    result    was    then  tested  by  Sternberg's  method    as  follows  : 

I  cc.  of  culture  was  introduced  into  i  cc.  of  disinfectant.  At  the  end  of  lo  seconds 
a  little  of  the  mixture  was  carried  on  a  platinum  needle  and  mixed  with  25  cc.  of  bouillon. 
The  flask  remained  sterile  after  being  in  the  incubator  for  three  days.  At  the  end  of 
15  seconds  another  flask  was  sown  in  the  same  manner.     This  flask  also  remained  sterile. 

A  further  confirmation  was  secured  in  the  following  way  : 

I  cc.  of  culture  was  introduced  into  i  cc.  of  disinfectant.  At  the  end  of  10  seconds  a  plati- 
num loop  of  the  mixture  was  mixed  with  i  cc.  of  dilute  ammonium  sulphide,  and  after  30  sec- 
onds here  the  mixture  was  poured  into  a  flask  of  bouillon  containing  25  cc.  The  bouillon 
flask  at  the  end  of  24  hours  contained  a  fine  crop  of  typhoid  bacilli.  At  the  end  of  15  seconds' 
exposure  to  the  disinfectant  a  second  platinum  loop  of  the  mixture  was  precipitated  in  the 
same  manner  and  sown  in  bouillon.     This  flask  remained  sterile. 

Conclusion  :  The  typhoid  fever  bacillus  of  Eberth  is  killed  by  an  exposure  of  15  seconds 
to  the  disinfectant  when  the  latter  is  diluted  to  twice  its  bulk. 


15 

B.     Experiments  tipon  Spores  of  Bacillus  Siibtilis. 

In  all  experiments  upon  Bacilhis  subtilis  we  employed  a  bouillon 
culture  grown  for  three  days  at  37.5°  C.  and  microscopically  determined 
to  be  in  the  spore  state. 

EXPERIMENT    I  5. 

2  cc.  of  culture  of  B.  siibtilis  mixed  well  with  13  cc.  of  sterile  water.  15  cc.  of  disinfectant 
then  poured  into  this.     Shaken  well  for  5  minutes. 

1  cc.  carried  to  500  cc.  of  ammonium  sulphide.  Left  here  for  30  seconds,  i  cc.  from 
Flask  2  then  carried  to  Flask  3,  containing  250  cc.  of  sterile  water. 

Bouillon  flasks  were  sown  from  Flask  2,  and  agar  plates  from  Flask  3. 

Result:  Growth  in  bouillon  flasks.     Agar  plates  contained  an  average  of  5  colonies. 

EXPERIMENT    1 6. 

Identical  with  Experiment  15,  except  that  stannous  chloride  was  employed  as  inhibitor. 
Exposure  to  disinfectant  of  5  minutes.     Bouillon  flasks  and  agar  plates  as  usual. 

Result:  Growth  in  bouillon  flasks.     Agar  plates  had  an  average  of  7  colonies  each. 

EXPERIMENT    I7. 

Identical  with  15.    Ammonium  sulphide,  inhibitor.    Exposure  of  7  minutes  to  disinfectant. 

Agar  plates  from  Flask  3,  bouillon  flasks  from  Flask  2. 

Result :  Bouillon  showed  a  copious  growth.    Agar  plates  an  average  of  8  colonies. 

EXPERIMENT    l8. 

Same  culture  as  preceding.  Stannous  chloride,  inhibitor.  Exposure  to  disinfectant  for 
7  minutes. 

Result :  Growth  in  bouillon  flasks.     Agar  plates  an  average  of  6  colonies. 

EXPERIMENT    I9. 

Identical  with  17,  except  that  exposure  was  for  10  minutes.  Ammonium  sulphide, 
inhibitor. 

Result :  Growth  in  bouillon  flasks.     Agar  plates  an  average  of  3  colonies. 

EXPERIMENT    20. 

Identical  with  i8,  except  exposure  of  lo  minutes.     Stannous  chloride,  inhibitor. 
Result :  Growth  in  bouillon  flasks.     Agar  plates  an  average  of  3  colonies. 

EXPERIMENT    21. 

Control  for  experiments  from  15-20. 

2  cc.  of  culture.     28  cc.  of  water. 

I  cc.  of  this  then  diluted  with  500  cc.  Ammonium  sulphide. 

I  cc.  of  mixture  then  carried  to  250  cc.  water. 

Result:  Agar  plates  showed  an  average  of  12  colonies. 

EXPERIMENT   22. 

Identical  with  17.     Exposure  for  \2.\  minutes.     Inhibitor,  ammonium  sulphide. 
Result:  Growth  in  bouillon  flasks.     Agar  plates  sterile. 


i6 

EXPERIMENT    23. 

Same  as  preceding.     Exposure  of  15  minutes.     Inhibitor,  ammonium  sulphide. 
Result :  Bouillon  flasks  sterile.     Agar  plates  sterile. 

EXPERIMENT   24. 

Control  for  22  and  23. 

2  cc.  culture.     28  cc.  water. 

I  cc.  of  mixture  carried  to  500  cc.  Ammonium  sulphide. 

I  cc.  of  this  to  250  cc.  water. 

Result :  Agar  plates  an  average  of  44  colonies. 

It  having  appeared  from  the  above  that  B.  subtilis  spores  are 
destroyed  in  fifteen  minutes  the  result  was  confirmed  by  Sternberg's, 
method.     The  results  were  as  follows  : 

Flask  I.     Exposed  15  minutes  showed  a  growth. 
Flask  2.     Exposed  17  minutes  remained  sterile. 

We  also  employed  the  modification  of  this  method  already  no- 
ticed.    The  results  of  this  were  as  follows  : 

Flask  I.     Exposed  15^  minutes  and  inhibited  with  ammonium  sulphide.     Growth. 

Flask  2.     Exposed  17^  minutes  and  inhibited  with  ammonium  sulphide.     Growth. 

Flask  3.     Exposed  20  minutes  and  inhibited  with  ammonium  sulphide.     Sterile. 

Conclusion:  Bacillus  subtilis ^'^ox^^  are,  under  certain  conditions,  killed  by  the  germicide 
diluted  to  twice  its  bulk  in  20  minutes.  Under  other  conditions  they  appear  to  be  killed  ia 
17  minutes. 


C.     Experitnejits  tipon  the  Streptococcus  of  pseudo-Diphtheria. 

This  Streptococcus  has  been  repeatedly  isolated  from  the  mem- 
brane in  the  throats  of  patients  sick  with  scarlet  fever,  and  others- 
erroneously  supposed  to  have  true  diphtheria.  It  apparently  bears 
an  aetiological  significance  in  the  cases  of  such  membranous  in- 
flammations of  the  throat.  It  has  already  been  established  at  this- 
laboratory  that  this  Streptococcus,  believed  by  some  to  be  identical 
with  Streptococcus  pyogenes,  is  even  more  resistant  to  germicides, 
than  the  bacillus  of  true  diphtheria,  i.e.,  the  bacillus  of  Klebs-Loffler. 


EXPERIMENT    25. 

2  CC.  culture,  13  cc.  water,  and  15  cc.  of  disinfectant,  mixed  for  10  seconds. 
I  cc.  of  the  mixture  then  mixed  with  500  cc.  of  ammonium  sulphide  for  30  seconds. 
I  cc.  of  this  mixture  transferred  to  Flask  3,  containing  250  cc.  water.     Bouillon  flasks, 
planted  from  Flask  2.     Agar  plates  with  i  cc.  each  from  Flask  3. 
Result :  Agar  plates  sterile.     Bouillon  showed  a  growth. 


17 


EXPERIMENT    26. 


Same  as  25,  except  the  exposure  of  12  seconds.     Ammonium  sulphide 
Result :  Agar  plates  sterile  ;  bouillon  flasks  sterile. 


EXPERIMENT    27. 

Control  of  25  and  26. 

2  cc.  of  culture  mixed  with  28  cc.  of  water. 

I  cc.  of  mixture  to  500  cc.  of  ammonium  sulphide  for  30  seconds. 

I  cc.  of  sulphide  flask  then  transferred  to  250  cc.  of  water. 

Agar  plates  from  Flask  3. 

Result :  Plates  showed  an  average  of  39  colonies. 


From  the  above  it  appeared  certain  that  an  exposure  for  twelve 
seconds  to  the  disinfectant  when  the  latter  is  diluted  to  twice  its 
volume  is  sufficient  to  kill  the   Streptococacs  diphtherice. 

This  result  was  confirmed  by  Sternberg's  method  and  the  modi- 
fication thereof  employed  in  the  case  of  B.  siibtilis  and  th&  Eberth 
bacillus. 

D.      Experiments  upon  Staphylococcus  pyogenes  mireus. 

The  culture  for  these  experiments  came  originally  from  the  labora- 
atory  of  Dr.  Councilman.     Three-days-old  bouillon  cultures  were  used. 

EXPERIMENT    28. 

2  cc.  bouillon  culture,   13  cc.  water,  and  15  cc.  disinfectant.     Exposed  10  seconds. 

I  cc.  of  mixture  then  carried  to  Flask  2,  containing  500  cc.  Ammonium  sulphide.  Ex- 
posed here  for  30  seconds. 

I  cc.  than  transferred  from  Flask  2  to  Flask  3,  containing  250  cc.  sterile  water. 

Bouillon  flasks  containing  25  cc.  bouillon  were  planted  with  .2  cc.  of  mixture  from  Flask  2. 
Agar  plates  with  i  cc.  each  from  Flask  3. 

Result:  Agar  plates  sterile.     Bouillon  flasks  sterile. 


EXPERIMENT   29. 

Same  as  above,  but  exposed  to  disinfectant  for  20   seconds.     Ammonium   sulphide  as 
inhibitor. 

Result .  Agar  plates  sterile.     Bouillon  flasks  sterile. 


EXPERIMENT   30. 

Control  for  28  and  29. 

2  cc.  culture  mixed  with  28  cc.  water. 

I  cc.  then  to  500  cc.  ammonium  sulphide. 

I  cc.  of  this  then  diluted  250  times  with  water. 

Result :  Agar  plates  from  last  flask  showed  530  colonies  each. 


i8 

The  conclusion  from  these  experiments  is  that  Staphylococcus  pyog- 
enes atcretis  is  killed  by  a  ten  seconds'  exposure  to  the  germicide  when 
the  latter  is  diluted  to  twice  its  bulk. 

This  result  was  confirmed  by  Sternberg's  method  and  the  modifi- 
cation of  it  already  described.  In  each  case  the  flasks  of  ten  seconds 
remained  sterile,  as  did  also  those  exposed  for  twelve  and  a  half 
seconds. 

E.     Experi'ments  upon  the  Sph'illmn  of  Asiatic  Cholera. 

For  these  experiments  we  used  a  bouillon  culture  grown  for  four 
days  at  37.5°  C.  The  culture  came  originally  from  Dr.  Dunham's 
laboratory. 

EXPERIMENT   3I. 

2  cc.  of  culture,  15  cc.  of  disinfectant,  and  13  cc.  of  sterile  water  were  mixed  for  10  seconds. 
I  cc.  of  mixture  precipitated  in  500  cc.     Ammonium  sulphide.     30  seconds. 

1  cc.  of  this  flask  then  carried  to  250  cc.  of  sterile  water. 

Bouillon  flasks  were  planted  from  Flask  2.     Agar  plates  from  Flask  3. 
Results :  Agar  plates  sterile.     Flasks  sterile. 

EXPERIMENT   32. 

Same  as  31,  except  the  exposure  to  the  disinfectant  was  for  15  seconds. 
Result :  Agar  plates  sterile.     Bouillon  flasks  sterile. 

EXPERIMENT   33. 

Control  for  31  and  32. 

2  cc.  of  culture  diluted  with  28  cc.  water,  then  i  cc.  passed  through  same  amounts  of 
ammonium  sulphide  and  water  as  usual. 

Agar  plates  were  planted  from  Flask  3. 

Result :  Agar  plates  showed  an  average  of  5  colonies  to  a  cubic  centimeter  of  liquid  from 
last  flask. 

The  dilutions  here  were  evidently  a  little  too  great,  but  when  it  is 
remembered  that  on  this  basis  the  middle  flask  should  contain  1,250 
cholera  bacilli  to  the  cubic  centimeter,  if  they  had  not  been  killed,  we 
considered  the  fact  of  the  sterility  of  the  flasks  of  bouillon  planted 
from  this  flask  as  evidence  enough  that  the  bacteria  were  killed  by 
the  exposure  given  them. 

The  experiments  upon  the  Spirillum  of  Asiatic  cholera  would  indi- 
cate that  this  bacterium  is  killed  by  an  exposure  to  the  germicide 
diluted  to  twice  its  volume  for  the  space  of  ten  seconds. 

This  result  was  confirmed  by  Sternberg's  method  and  the  modifica- 
tion of  it  described.     Bouillon  flasks  at  10  seconds  remained  sterile. 


19 

F.     Experiments  7ipon  Spores  of  Bacillus  anthracis. 

The  culture  of  Bacilhis  anthracis  used  in  these  tests  was  from  a 
luxuriant  culture  isolated  by  Prof.  G.  R.  Tucker  from  a  patient  in  the 
Boston  City  Hospital  about  one  year  before.  A  three-days'  bouillon 
culture  was  used. 


EXPERIMENT   34. 

2  cc.  of  culture  well  shaken  in  13  cc.  of  water;  15  cc.  of  disinfectant  mixed  with  it  tor 
15  minutes. 

I  cc.  of  the  mixture  was  then  inhibited  by  500  cc.  of  ammonium  sulphide. 

1  cc.  from  the  last  flask  was,  at  the  end  of  30  seconds,  carried  to  Flask  3,  containing 
250  cc.  of  distilled  water. 

Agar  plates  made  from  the  last  flask.     Bouillon  flasks  planted  from  the  inhibition  flask. 
Result :  Agar  plates  had  an  average  of  3  colonies  each.     Bouillon  flasks  showed  copious 
growth. 

EXPERIMENT   35. 

Control  for  34. 

2  cc.  of  culture  mixed  with  28  cc.  of  water. 

I  cc.  of  mixture  then  transferred  to  500  cc.  of  ammonium  sulphide  for  30  seconds. 

I  cc.  of  mixture  then  diluted  in  250  cc.  of  sterile  water. 

Agar  plates  from  last  flask. 

Result :   Plates  contained  an  average  of  52  colonies  each. 

We  next  passed  on  to  determine  the  vitality  of  the  spores  and 
found  them  exceedingly  resistant  to  heat. 

EXPERIMENT   36. 

I  cc.  of  the  culture  used  in  the  preceding  two  experiments  was  placed  in  a  sterile  test 
tube  and  immersed  in  boiling  water.  Every  two  minutes  a  sterile  platinum  needle  was 
plunged  into  the  hot  culture  and  then  mixed  with  6  cc.  of  sterile  bouillon.  In  order  to  deter- 
mine the  exact  degree  of  heat  to  which  the  spores  were  exposed  we  placed  next  to  the 
tube  containing  the  culture  a  second  tube  containing  i  cc.  of  sterile  water,  and  within 
this  tube  suspended  a  thermometer  so  that  it  was  at  every  point  separated  from  the  glass  of 
the  tube.  This  thermometer  registered  as  follows  :  5  seconds,  60  degrees  C. ;  10  seconds,  70 
degrees;  15  seconds,  80  degrees;  30  seconds,  92  degrees;  45  seconds,  95  degrees  ;  60  seconds, 
97  degrees ;  70  seconds,  99  degrees.  It  remained  at  this  temperature  during  the  course  of 
the  tests. 

The  result  proved  that  some  of  the  spores  were  still  alive  at  eleven 
minutes  after  immersion.     We  made  no  test  later  than  that  time. 

Experiment  34  indicated  that  nearly  all  the  spores  were  killed  after 
fifteen  minutes'  exposure  to  the  disinfectant. 

EXPERIMENT   37. 

Identical  with  34,  except  that  the  exposure  was  for  20  minutes. 
Result :  Agar  plates  sterile.     Bouillon  flasks  sterile. 


20 


Having  now  ascertained  that  anthrax  spores  of  a  very  resistant 
type  are  killed  by  the  disinfectant  in  twenty  minutes,  we  confirmed 
the  result  as  usual,  by  Sternberg's  method,  and  also  by  the  modifica- 
tion of  that  method  already  described. 

It  will  be  remembered  that  in  the  modification  of  the  method  we 
precipitated  a  platinum  loopful  of  the  mixture  of  culture  and  disin- 
fectant in  ammonium  sulphide  before  pouring  the  whole  mixture  into 
bouillon. 

Result : 


li  minutes    .     . 

.     Direct. 

Growth. 

14    minutes  .     . 

.     Direct. 

Growth. 

3  minutes    .     . 

.     Direct. 

Growth. 

17    minutes  .     . 

.     Precipitated. 

Growth 

5  minutes    . 

.     Direct. 

Growth. 

19    minutes  .     . 

.     Direct. 

Sterile. 

8  minutes    .     . 

Precipitated. 

Growth. 

17^  minutes  .     . 

.     Precipitated. 

Growth 

lo  minutes    . 

.     Direct. 

Growth. 

19    minutes  .     . 

.     Precipitated. 

Sterile. 

12  minutes    .     . 

.     Precipitated. 

Growth. 

From  this  result  we  may  conclude  that  anthrax  spores  of  this  high 
degree  of  resistance  are  killed  by  an  exposure  to  the  disinfectant  of 
nineteen  minutes,  the  disinfectant  being  diluted  to  twice  its  volume. 


G.     A  Practical  Test  of  the  Disinfectant. 

In  order  to  test  the  working  of  the  disinfectant  in  actual  use  we 
mixed  a  stool  of  a  typhoid  patient  with  an  equal  amount  of  disinfect- 
ant. The  stool  was  diarrhoeic,  but  contained  many  hard  lumps  of 
fasces.  The  mixture  was  shaken  three  or  four  times  during  the  test. 
In  order  to  determine  at  what  time  the  stool  became  sterile  we  trans- 
ferred a  small  portion  upon  a  platinum  loop  at  the  end  of  thirty  sec- 
onds, and  for  every  minute's  interval  thereafter  up  to  twenty  minutes, 
to  a  tube  of  melted  agar  containing  perhaps  6  cc.  of  agar.  At  the  end 
of  twenty-four  hours  the  plates  planted  at  thirty  seconds'  exposure 
proved  to  be  well  spotted  with  colonies  of  bacteria,  but  far  less  than 
the  control  made  at  the  beginning.  From  this  time  on  they  rapidly 
diminished  until  at  the  end  of  three  minutes  and  a  half  there  were 
none  apparent,  nor  did  any  appear  upon  the  subsequent  plates  or  in 
the  bouillon  flasks.  The  efficiency  of  the  disinfectant  with  faecal 
bacteria  was  thus  established. 


21 


SUMMARY   OF   EXPERIMENTS   ON   B.    TYPHI  ABDOMINALTS. 


No.  of  the 
Experiment. 

Age  of 
Culturft. 

Strength 
ot  Dis- 
infectant. 

Time  of  Ex- 
posure to 
Disinfectant 

Precipitant 
used  as 
Inhibitor. 

Colonies  on 
Agar  Plates. 

Bouillon 
Flasks. 

Number  of 

Colonies  on 

Control. 

1. 

50  days 

I 

1  minute 

SnCl, 

3 

•    •    . 

2. 

50     " 

\ 

1       " 

HoO 

0 

3. 

1\     " 

i 

20  seconds 

(NH4)oS 

0  and  1 

11 

4. 

1\     " 

i 

18        <' 

H2O 

0  and  1 

11 

5. 

2\     " 

not  exposed 

SnCl2 

11 

7. 

2      " 

i 

25  seconds 

SnCIa 

0 

2035 

8. 

2      " 

i 

45       " 

SnCl2 

0 

2035 

10. 

2      " 

\ 

10       " 

SnClo 

0and2 

growth. 

150 

11. 

2      " 

\ 

15       " 

SnCU 

0 

sterile. 

150 

12. 

2      " 

\ 

20       " 

SnCl. 

0 

" 

150 

13. 

2      « 

\ 

15       " 

(NH4)2S 

0 

a 

150 

14. 

2      " 

\ 

10      " 

(NH4)2S 

0 

" 

150 

Control:  Sternberg's  Method,  Flask  at  10  seconds,  sterile. 

Control:  Modification  of  Sternberg's  Method,  Flask  at  10 seconds,  growth;  15  seconds, 
sterile. 


SUMMARY   OF   EXPERIMENTS   ON   SPORES   OF  BACILLUS  SUB  TILLS. 


No.  of  the 
Experiment. 

Age  of 
Culture. 

Strength 
of  Dis- 
infectant. 

Time  of  Ex- 
posure to 
Disinfectant. 

Precipitant 

used  as 
Inhibitor. 

Colonies  on 
Agar  Plates. 

Bouillon 
Flasks. 

Number  of 

Colonies  on 

Control. 

15. 

3   days 

i 

5  minutes 

{NH4)2S 

5 

growth 

12 

16. 

3      " 

1 

5       " 

SnCl2 

7 

12 

17. 

3      " 

i 

7       " 

(NH4)2S 

8 

12 

18. 

3      " 

I 

7       " 

SnCla 

6 

12 

19. 

3       " 

1 

2 

10       " 

(NH4)2S 

3 

12 

20. 

3       " 

\ 

10       " 

SnCl2 

3 

12 

22. 

3       " 

k 

12^     " 

(NH4)2S 

0 

44 

23. 

3       " 

\ 

15       " 

(NH4)2S 

0 

sterile 

44 

Control :  Sternberg's  Method,  Flask  at  15  minutes,  growth  ;  17  minutes,  growth. 
Control :  Sternberg's  Method,  modified.  Flask  at  15  minutes,  growth;  17  minutes,  growth ; 
20  minutes,  sterile. 


22 


SUMMARY   OF  EXPERIMENTS   ON   STREPTOCOCCUS  DIPHTHERIA. 


No.  of  the 
Experiment. 

Age  of 
Culture. 

Strength 
of  Dis- 
infectant. 

Time  of  Ex- 
posure to 
Disinfectant. 

Precipitant 

used  as 
Inhibitor. 

Colonies  on 
Agar  Plates. 

Bouillon 
Flasks. 

Number  of 

Colonies  on 

Control. 

25. 
26. 

3   days 
3       " 

10  seconds 
12       " 

(NH4)2S 
(NH4)2S 

0 
0 

growth 
sterile 

39 
39 

Control :  Sternberg's  Method,  Flask  at  10  seconds,  growth ;   12  seconds,  sterile. 

Control :  Sternberg's  Method,  modified,  Flask  at  10  seconds,  growth ;   12  seconds,  sterile. 

SUMMARY   OF  EXPERIMENTS   ON   S.  PYOGENES  AUREUS. 


No.  of  the 
Experiment. 

Age  of 
Culture. 

Strens;th 
of  Dis- 
infectant. 

Time  of  Ex- 
posure to 
Disinfectant. 

Precipitant 
used  as 
Inhibitor. 

Colonies  on 
Agar  Plates. 

Bouillon 
Flasks. 

Number  of 

Colonies  on 

Control. 

28. 
29. 

3    days 
3       " 

10  seconds 
20       " 

(NH4)2S 
(NH4)2S 

0 
0 

Sterile 
sterile 

530 
530 

Control :   Sternberg's  Method,  Flask  at  10  seconds,  sterile ;  at  12  seconds,  sterile. 
Control :  Sternberg's  Method,  modified.  Flask  at  10  seconds,  sterile  ;  at  12  seconds,  sterile. 

SUMMARY   OF   EXPERIMENTS   ON   SPIRILLUM   OF  ASIATIC    CHOLERA. 


No.  of  the 
Experiment. 

Age  of 
Culture. 

Strength 
of  Dis- 
infectant. 

Time  of  Ex- 
posure to 
Disinfectant. 

Precipitant 
used  as 
Inhibitor. 

Colonies  on 
Agar  Plates. 

Bouillon 
Flasks. 

Number  of 

Colonies  on 

Control. 

3L 
32. 

4   days 
4       " 

1 
2 

10  seconds 
15       " 

(NH4)2S 
(NH4)2S 

0 
0 

sterile 
sterile 

5 
5 

Control:  Sternberg's  Method,  10  seconds  flask,  sterile. 

Control :  Sternberg's  Method,  modified,  10  seconds  flask,  sterile. 


SUMMARY  OF  EXPERIMENTS  ON   SPORES   OF  BACILLUS  ANTHRACIS. 


No.  of  the 
Experiment. 

Age  of 
Culture. 

Strength 
of  Dis- 
infectant. 

Time  of  Ex- 
posure to 
Disinfectant. 

Precipitant 

used  as 
Inhibitor. 

Colonies  on 
Agar  Plates. 

Bouillon 
Flasks. 

Number  of 

Colonies  on 

Control. 

34. 

37. 

3    days 
3      " 

15  minutes 
20       " 

(NH4)2S 
(NH4)2S 

3 
0 

growth 
sterile 

52 
52 

Control :  Sternberg's  Method,  Flask  at  19  minutes,  sterile. 

Control :  Sternberg's  Method,  modified.  Flask  at  17  minutes,  growth  ;   19  minutes,  sterile. 


23 


CONCLUSIONS. 


The  foregoing  experiments  appear  to  establish  the  fact  that  the 
Standard  Disinfectant  used  by  the  Pennsylvania  Railroad  Company 
will,  under  favorable  circumstances,  destroy  the  germs  of  typhoid  fever, 
diphtheria,  Asiatic  cholera,  and  Staphylococcus  pyogenes  aureus  in  a  few 
seconds,  and  the  spores  of  Bacillus  subtilis  and  Bacillus  anthracis  in 
a  few  minutes.  It  must,  therefore,  be  regarded  as  satisfactory  and  of 
excellent  efficiency. 

There  is  no  evidence  that  the  zinc  chloride  materially  contributes 
to  its  value  as  a  disinfectant,  and  however  great  the  value  of  zinc 
chloride  as  a  deodorant  may  be,  it  is  probable  that  if  a  portion,  or  all 
of  this,  could  be  replaced  by  a  still  more  concentrated  solution  of 
corrosive  sublimate  the  disinfectant  might  be  improved.  The  recent 
paper  of  Green  {Zeit.  fiir  Hygiene,  etc.,  13,  511)  suggests  that  the 
copper  chloride  also  has  some  value.  Finally,  the  decidedly  acid  re- 
action of  the  disinfectant  as  received  is,  from  the  germicidal  point 
of  view,  an  advantage. 


COLUMBIA  UNIVERSITY  LIBRARIES 

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DATE  BORROWED 


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2a(1  140)M100 


GAYLAMOUNT 
PAMPHLET  BINDER 

Manu/acluted  by 

1  GAYLORD  BROS.  Inc. 

Syracuse,  N.  Y. 

Slocklon,  Calif. 


RA615 
Sedgwick 


Se2 


Se.Z 


